SUMMARY In humans, there are over 460 Solute Carrier (SLC) transporters that mediate the import and efflux of solutes, including neurotransmitters, nutrients, and ions. The SLCs are emerging as key therapeutic targets for a variety of diseases and disorders. For example, nutrient SLC transporters play a major role in reprogrammed metabolic networks in cancer, autoimmune disease, and heart ischemia, by supplying cells with nutrients that are used to build biomass or serve as signaling molecules that enhance cell proliferation and differentiation, or regulate cell death. Our broad goal is to describe the substrate and inhibitor specificity determinants in nutrient transporters and develop novel strategies to modulate their functions. We take an integrative approach that includes computational biology and organic chemistry, coupled with biochemical and biophysical approaches, and in vitro models of disease, to characterize the Alanine-Serine-Cysteine Transporter 2 (ASCT2, SLC1A5). In Aim 1 of this project, we will generate and refine homology models for the human SLC1 family in multiple conformations. We will describe structural features of the models? substrate binding site and recently discovered allosteric site(s), that determine substrate and inhibitor specificity. Key residues will be tested with site-directed mutagenesis and electrophysiological approaches. Finally, we will develop a publicly-accessible database that includes homology models for all modelable human SLC transporters, and maps disease-related mutations onto the models and provides their predicted functional effect. In Aim 2, we will rationally design small molecule tool compounds that modulate the function of nutrient transporters via distinct mechanisms. This will expand the chemical space of transporter inhibitors and provide a test for the models developed in Aim 1. We will focus on the following approaches: (A) optimizing and characterizing novel conformation-specific inhibitors, such as photoactivatable and fluorescent compounds that interact with the substrate binding site of ASCT2; (B) designing allosteric inhibitors targeting the domain-domain interface in ASCT2, to test the putative elevator mechanism of this transporter; and (C) developing inhibitors with dual specificity for ASCT2 and the L-type Amino Acid Transporter 1 (LAT1, SLC7A5), which we hypothesize to have a pronounced inhibitory effect on cell proliferation due to their cooperativity in reprogrammed metabolic networks. If successful, this project will improve our understanding of structure-function relationships in emerging therapeutic targets. This project will also provide novel chemical probes to further characterize the structure of these proteins and their role in disease, as well as a useful publicly available online resource.